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Molecular Microbiology (2010) 77(1), 252–271 䊏 doi:10.1111/j.1365-2958.2010.07206.x First published online 7 June 2010 The twin arginine protein transport pathway exports multiple virulence proteins in the plant pathogen Streptomyces scabies mmi_7206 252..271 Madhumita V. Joshi,1† Stefan G. Mann,2† Haike Antelmann,3 David A. Widdick,4,5 Joanna K. Fyans,4,6 Govind Chandra,2 Matthew I. Hutchings,5 Ian Toth,6 Michael Hecker,3 Rosemary Loria1 and Tracy Palmer4* 1 Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY 14853, USA. 2 Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK. 3 Institute for Microbiology, Ernst-Moritz-Arndt-University of Greifswald, D-17487 Greifswald, Germany. 4 Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK. 5 School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK. 6 Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK. Summary Streptomyces scabies is one of a group of organisms that causes the economically important disease potato scab. Analysis of the S. scabies genome sequence indicates that it is likely to secrete many proteins via the twin arginine protein transport (Tat) pathway, including several proteins whose coding sequences may have been acquired through horizontal gene transfer and share a common ancestor with proteins in other plant pathogens. Inactivation of the S. scabies Tat pathway resulted in pleiotropic phenotypes including slower growth rate and increased permeability of the cell envelope. Comparison of the extracellular proteome of the wild type and DtatC strains identified 73 predicted secretory proteins that were present in reduced amounts in the tatC mutant strain, and 47 Tat substrates were verified using a Tat reporter assay. The DtatC strain was almost completely avirulent on Arabidopsis seedlings and was delayed in attaching to the root tip relative to the Accepted 4 May, 2010. *For correspondence. E-mail t.palmer@ dundee.ac.uk; Tel. (+44) (0)1382 386464; Fax (+44) (0)1382 388216. † These authors contributed equally to this work. © 2010 Blackwell Publishing Ltd wild-type strain. Genes encoding 14 candidate Tat substrates were individually inactivated, and seven of these mutants were reduced in virulence compared with the wild-type strain. We conclude that the Tat pathway secretes multiple proteins that are required for full virulence. Introduction Potato scab is a polyphyletic disease caused by organisms in the genus Streptomyces, the best studied of which is Streptomyces scabies (Loria et al., 2006). Like other streptomycetes, S. scabies is a mycelial bacterium that undergoes complex morphological differentiation involving the formation of aerial hyphae and spores (see Flärdh and Buttner, 2009 for a review of the complex biology of Streptomyces). The hyphal form of S. scabies infects plants through expanding plant tissue, including roots and tubers (Loria et al., 2006; 2008). One of the major pathogenicity determinants of S. scabies is thaxtomin, a nitrated dipeptide toxin that is a potent inhibitor of cellulose synthesis. The toxin induces plant cell hypertrophy in expanding plant tissues and likely facilitates penetration of plant tissue by the pathogen (King et al., 1989; Healy et al., 2000; Scheible et al., 2003; Bischoff et al., 2009). The mechanism by which thaxtomin inhibits cellulose synthesis is undefined but it apparently interacts with a highly conserved target, as the toxin affects all higher plants; this is congruent with the fact that thaxtomin-producing streptomycetes have an extremely wide host range. The thaxtomin biosynthesis genes are conserved in the plant pathogenic species, including Streptomyces turgidiscabies and Streptomyces acidiscabies, and are regulated by TxtR, an AraC family transcriptional regulator that binds cellobiose as a co-inducer (Kers et al., 2005; Joshi et al., 2007a). The S. scabies 87-22 genome contains a biosynthetic cluster that is highly similar in structure and organization to the coronafacic acid (CFA) clusters from the Gram negative plant pathogens Pseudomonas syringae, and Pectobacterium atrosepticum (Bignell et al., 2010). CFA is the polyketide component of coronatine that acts as a jasmonate mimic during plant interactions; mutational studies demonstrate The Tat pathway in S. scabies 253 that the cluster contributes to virulence in S. scabies 87-22 as it does in Gram negative pathogens. Unlike the thaxtomin biosynthetic cluster, the coronafacic acid-like cluster is not conserved in S. turgidiscabies or S. acidiscabies. Secreted proteins are also critical determinants in the interaction between bacteria and eukaryotic hosts; virulence proteins are secreted either into the host environment or directly into host cells. Indeed, a secreted proteinaceous virulence factor, Nec1, lacks homologues outside of plant pathogenic streptomycetes, is required for colonization of the roots and may function to suppress plant defence responses (Bukhalid and Loria, 1997; Joshi et al., 2007b). Furthermore, the secreted protein TomA, is conserved among pathogenic streptomycetes (Kers et al., 2005) and is homologous to products of saponinaseencoding genes, which are important for host–pathogen interactions in some plant pathogenic fungi (Seipke and Loria, 2008). The role of protein secretion in pathogenesis is particularly well characterized in the case of Gram negative bacteria, which have numerous systems dedicated to achieving the secretion of proteins across the complex double-membrane cell envelope (see, e.g. Christie et al., 2005; Büttner and He, 2009; Galán, 2009). In contrast, Gram positive bacteria have a simpler cell envelope, and secretion generally requires passage of proteins across only a single membrane. Therefore, it might be expected that the general protein transport machineries residing in the prokaryotic cytoplasmic membrane play more direct roles in the virulence of Gram positive organisms. In support of this it has been shown that many Gram positive bacteria have an accessory SecA protein, termed SecA2 that appears to contribute to bacterial virulence (e.g. Rigel and Braunstein, 2008). The Tat system is, like Sec, a general protein export pathway that is found in the cytoplasmic membranes of some (although not all) bacteria and archaea. In Gram negative bacteria and in the Gram positive Actinobacteria, that include the streptomycetes, the Tat system is comprised of three essential components, TatA, TatB and TatC (Bogsch et al., 1998; Sargent et al., 1998; 1999; Weiner et al., 1998; Schaerlaekens et al., 2004; Hicks et al., 2006). By contrast, the Tat machineries in the low G + C Gram positive bacteria (exemplified by Bacillus subtilis) and in the archaea do not require a TatB protein and the Tat systems in these prokaryotes are made up only of TatA and TatC components (Jongbloed et al., 2004; Dilks et al., 2005). Proteins are targeted to the Tat pathway by means of N-terminal signal peptides that superficially resemble Sec signal peptides, but that harbour a conserved S/T-R-R-xF-L-K consensus motif, where the twin arginines are invariant and normally essential for efficient export by the Tat pathway (Berks, 1996; Stanley et al., 2000). The main distinguishing feature of the Tat system is that it transports fully folded proteins across the cytoplasmic membrane. In bacteria such as Escherichia coli and B. subtilis, relatively few proteins are exported by the Tat pathway and, in E. coli at least, the majority of them contain complex redox cofactors that are assembled into the substrate protein prior to transport across the membrane (reviewed in Palmer et al., 2005; Lee et al., 2006). Although in most organisms the Tat pathway is generally held to be a relatively minor route of protein export, there is evidence that in some halophilic archaea, and in particular in Streptomyces bacteria, Tat exports significant numbers of proteins. Thus, bioinformatic predictions on the genome sequence of Streptomyces coelicolor suggested that as many as 189 proteins may be substrates of the Tat pathway (Rose et al., 2002; Dilks et al., 2003; Bendtsen et al., 2005). The validity of these prediction programmes were largely borne out by proteomic studies of S. coelicolor wild type (WT) and tat mutant strains coupled with testing of candidate Tat signal peptides in Tat-dependent reporter assays, where a total of 33 Tat substrate proteins have now been confirmed (Li et al., 2005; Widdick et al., 2006). In spite of the fact that the Tat system is a general protein export system, Tat-secreted proteins contribute to virulence in some Gram negative and Gram positive bacteria (e.g. Ochsner et al., 2002; Ding and Christie, 2003; Pradel et al., 2003; Saint-Joanis et al., 2006). Given the apparent importance of the Tat pathway in protein secretion in the streptomycetes, we reasoned that this pathway may play an essential role in the virulence of S. scabies. Analysis of the recently available S. scabies 87-22 genome sequence (http://www.sanger. ac.uk/Projects/S_scabies/) with Tat signal peptide prediction programmes suggests that in excess of 100 proteins may be exported by the Tat pathway in this organism, including several that appear to share a common ancestor with homologues in other plant pathogens. Through proteomic studies coupled with reporter protein secretion assays we have verified 47 Tat substrates in this organism. Importantly, we show that a DtatC mutant of S. scabies is almost completely avirulent, and that the Tat pathway secretes multiple proteins that are required for full virulence. Results Bioinformatic analysis of the S. scabies genome sequence indicates that it encodes many candidate Tat substrates Two generally available prediction programmes, TATFIND 1.4 and TatP, have been developed to identify candidate Tat-targeting signal peptides (Rose et al., © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 254 M. V. Joshi et al. 䊏 2002; Bendtsen et al., 2005). When these programmes are applied to all of the open reading frames (ORFs) encoded by the genome sequence of S. scabies, TATFIND 1.4 predicts 154 likely Tat substrates, while TatP predicts 177 (the list of these proteins is available at http://www.lifesci.dundee.ac.uk/groups/tracy_palmer/ links.html). However, both of these programmes generate a degree of false positive and false negative predictions; for example, when applied to the genome sequence of E. coli, both programmes overpredict Tat substrates by 20–25% (Dilks et al., 2003; Bendtsen et al., 2005). In general, the sets of candidate Tat substrates predicted by the two programmes show partial but not complete overlap. However to date, where tested, all of those predicted to be Tat substrates by both TATFIND 1.4 and TatP have been shown to have bona fide Tat-targeting signals (Widdick et al., 2006). This therefore gives confidence that the subset of proteins predicted to be Tat substrates by both programmes are highly likely to represent real substrates. For S. scabies, 82 proteins are predicted to be Tat substrates by each of the two programmes, and these are listed in Table S1. It was noted from previous work with S. coelicolor that it is sometimes difficult to identify the correct N-terminus of a predicted protein and this can lead to mis-annotation of start codons (D.A. Widdick, G. Chandra and T. Palmer, unpublished). Therefore, all of the ORFs of S. scabies were re-analysed to take account of all potential start codons; these modified ORFs were also analysed by TATFIND 1.4 and TatP, leading to the identification of a further 21 potential Tat substrates, which are also listed in Table S1. Additionally, it is known that some Tat signals have very long n-regions that preclude their identification by TATFIND 1.4 or TatP; these programmes have maximum preferred lengths for signal peptide n-regions. Therefore all of the S. scabies ORFs were truncated in silico by 30 or 60 amino acids and re-analysed by TATFIND 1.4 and TatP resulting in the identification of an additional five candidate Tat substrates (Table S1). Finally, all of the ORFs that were predicted to have Tat signal peptides by only one of the two Tat signal peptide prediction programmes were sorted manually for those that were likely to be true Tat substrates on the basis of binding a complex cofactor, showing high homology to confirmed Tat substrates from other organisms, or by virtue of the fact that the twin arginine motif was highly conserved across bacterial homologues. This added a further 14 substrates to the manually curated list of likely Tat substrates (Table S1). An additional four proteins were added as a result of the outcome of the Tat-dependent reporter experiments described below and this curated list of likely Tat substrates in S. scabies (Table S1) therefore contains a total of 126 proteins. Phenotype of a S. scabies DtatC deletion strain In order to test the in silico predictions that there are many Tat substrates encoded in the genome of S. scabies, it was necessary to inactivate the Tat pathway. Inspection of the genome sequence of S. scabies reveals that, like other streptomycetes, it encodes the tatA (SCAB73591) and tatC (SCAB73601) genes in close proximity (separated by 51 bp) and a probable tatB gene (SCAB31121) at a distant location. Because the tatC gene encodes an essential component of the Tat transporter (e.g. Bogsch et al., 1998), we constructed a marked deletion of tatC as described in Experimental Procedures. Inactivation of the Tat pathway in S. coelicolor and Streptomyces lividans is associated with a number of phenotypic changes including a dispersed growth in liquid culture (rather than the mycelial pellets that are seen for the WT strains), failure to sporulate on solid media containing sucrose and increased fragility of the hyphae that may reflect a cell wall defect (Schaerlaekens et al., 2004; Widdick et al., 2006). To ascertain whether inactivation of the Tat pathway had similar pleiotropic effects on S. scabies, we initially assessed the growth rate of the S. scabies WT and isogenic DtatC strains. It should be noted that Streptomyces growth rates cannot be measured by spectroscopic analysis as the presence of mycelial clumps in liquid culture, coupled with the production of cellular debris by programmed cell death, distorts results obtained by spectroscopy (Miguélez et al., 1999). Therefore to assess growth, total cytosolic protein was extracted from living cells and measured, as described in Experimental Procedures. As shown in Fig. 1A, when cultured in tryptone soya broth (TSB) medium, the DtatC mutant strain does not grow as well as the WT strain. While both strains reach a peak of growth at around 28 h post inoculation (hpi), the mutant strain showed only about half of the total protein content per ml of culture as the WT strain. Following the growth peak, both strains showed a subsequent decline in the total protein content that may be related to programmed cell death (Miguélez et al., 1999). The slower growth rate of the DtatC strain was also apparent on solid growth media; for example, the tatC mutant strain formed smaller colonies on yeast extract malt extract (YEME) agar plates (Fig. 1B). It is also striking to note in Fig. 1B that the DtatC strain lacks the brown colouration of the medium and the hyphae that is associated with the WT strain. This may reflect a lack of production of melanin, which is catalysed by tyrosinase, a cofactor-containing Tat substrate (Schaerlaekens et al., 2001). Two probable tyrosinase enzymes are encoded in the S. scabies genome, SCAB85681/85691 and SCAB59231/59241, and the MelC1 components of both of these have candidate twin arginine signal peptides (see Table S1). It should be noted © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 The Tat pathway in S. scabies 255 Fig. 1. Inactivation of the Tat pathway in S. scabies results in pleiotropic growth phenotypes. A. 100 ml of TSB medium was inoculated with either S. scabies WT or DtatC strains, at a concentration of 100 000 spores per ml and incubated at 30°C with shaking. At the indicated time points, 3 ¥ 1 ml samples were removed from each culture, the hyphae pelleted and total protein content was determined using the Biorad DC Protein Assay. The error bars represent the standard error of the mean of samples taken at indicated time points, where n = 3. B. Spores of the indicated strains were streaked out from a fresh solid medium culture with an inoculation loop onto YEME medium and incubated for 7 days at 30°C. C. Spores dilutions of each strain were plated onto YEME medium and onto the same medium containing 0.01% SDS. The plates were incubated at 30°C for 7 days. D. Approximately 107 spores of each strain were spread on 144 cm2 DNA medium. Antibiotic discs with of 6 mm diameter were imbued with one of the following different amounts of vancomycin in mg as indicated and plates were incubated at 30°C for 7 days. that although the phytotoxin thaxtomin is also pigmented, thaxtomin production was not affected by inactivating tatC (Fig. S1). Complementation of the S. scabies DtatC strain with an integrative plasmid harbouring the S. scabies tatAC genes restored production of the brown pigment, and also reversed the slow growth rates seen on solid media, indicating that these phenotypes are directly linked to the tatC mutation (Fig. S2). Inactivation of the Tat pathway is often associated with a decrease in the integrity of the bacterial cell envelope, and in some organisms at least this is linked to an inability to export Tat-dependent proteins involved in cell wall remodelling (Ize et al., 2003; Caldelari et al., 2006). In E. coli and P. syringae this has been observed as an increased sensitivity to the presence of the detergent SDS when the Tat pathway is inactivated. As shown in Fig. 1C, deletion of the S. scabies tatC gene also results in an increased sensitivity to killing by SDS, which might also be suggestive of a Tat-linked cell envelope defect in this organism. A possible underlying defect in the S. scabies cell wall linked to inactivation of the Tat pathway was investigated by determining the sensitivity of the WT and DtatC strains to the cell wall-directed antibiotic vancomycin. As shown in Fig. 1D, the DtatC strain was clearly significantly more sensitive to killing by vancomycin than the WT strain, and a similar result was also seen with a different cell wall-directed antibiotic, bacitracin (data not shown). These observations suggest that the cell wall of the tatC mutant strain differs from that of the WT. © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 256 M. V. Joshi et al. 䊏 Fig. 2. Close-up of protein spots that are predominantly present in the WT extracellular proteome in all four types of growth media. The S. scabies WT and DtatC strains were grown on all four of the following growth media; IPM, OBM, R5 and SFM, and extracellular proteins were harvested from cell wall washes, TCA precipitated and subjected to 2D-PAGE as described in Experimental Procedures. Depicted are enlarged sections of the dual channel images of the extracellular proteomes of S. scabies WT (red colour) in comparison to the tatC mutant strain (green colour). The red-appearing proteins are labelled by the SCAB number and include 28 proteins that are strongly exported in more than one growth medium in the WT and decreased or absent in the DtatC mutant. The ratios for all identified proteins exported at lower amounts by the DtatC mutant are given in Table S2. Analysis of the exported proteome of the S. scabies WT and DtatC strains We next chose to examine the Tat-dependent proteome of S. scabies by comparison of the extracellular proteins produced by the WT and the tatC mutant strains under different growth conditions. As noted previously, the complex life cycle of streptomycetes results in significant lysis of the hyphae in liquid culture, and release of many cytoplasmic proteins into the growth medium. Therefore, we followed the procedure of Widdick et al. (2006), which involves growing Streptomyces on solid media on top of cellophane discs and washing the biomass with lithium chloride to release cell wall-associated proteins. The strains were cultured on four standard S. scabies growth media – oat bran medium (OBM), instant potato mash medium (IPM), soy-flour mannitol medium (SFM) and R5 medium (which is a more defined medium than the other three and lacks plant-derived material), and proteins from the cell wall washes were analysed by two-dimensional gel electrophoresis. For each growth medium analysed, the proteomes of the WT and tatC mutant strain were compared using the Decodon Delta 2D software with the WT proteins false coloured in red and the tatC proteins in green to allow differences to be visualized. The results of the proteome comparison for each type of growth media are shown in Fig. S3, and close-ups of some abundant red protein spots that are detected in the WT but are absent or decreased in the DtatC strain in at least two different growth media are presented in Fig. 2. It is clear from inspecting the gels shown in Fig. S3 and the panels in Fig. 2 that many proteins are exported at lower amounts in the tatC mutant strain. The redappearing protein spots were identified using tryptic © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 The Tat pathway in S. scabies 257 digestion and MALDI-ToF mass spectrometry. In total, 103 proteins were identified – these are indicated on Figs. 2 and S3, and are listed in Table 1. The ratios of the protein amounts for all red spots that are more highly exported in the WT were quantified in relation to the DtatC strain and presented in Table S2. The sequence coverages and protein scores for all identified proteins are given in Table S3. Among the 103 proteins we identified, 73 proteins were predicted to have N-terminal signal peptides. Of these, 14 were predicted to be Tat substrates by both TATFIND and TatP (and this was increased to 19 if alternative start sites were selected), and a further nine were predicted to be Tat substrates by one of the two programmes. Interestingly, these 28 predicted Tat substrates include several enzymes that are related to phosphate starvation conditions, such as alkaline phosphatases (SCAB77971, SCAB68191) glycerophosphoryl diesterase (SCAB74351), and 5′-nucleotidases (SCAB68841, SCAB49491), which are abundantly exported proteins in WT cells (Fig. 2). Furthermore, several glycosyl hydrolases (SCAB16771, SCAB17721), cellulases (SCAB25591) and mannosidases (SCAB16551) were exported strongly in WT cells but at significantly reduced levels in the tatC mutant strain. In addition to these 28 predicted Tat substrates, there were 50 signal peptide-bearing proteins that were reduced in the extracellular proteome of the tatC mutant but were not predicted to be Tat substrates and many of these did not have consecutive arginines in their signal peptides. It should be noted that the majority of exported proteins identified as being differentially missing from the extracellular proteome of the S. coelicolor DtatC strain were also not Tat substrates, and it was concluded that they were simply not produced in the DtatC strain because of the pleiotropic effects of the mutation (Widdick et al., 2006). It is possible that some of these differences also relate to the different growth rates of the S. scabies WT and DtatC strains. Finally, 30 proteins were identified in the cell wall wash of the S. scabies WT strain that did not appear to have N-terminal signal peptides (Table 1). It is likely that most of these proteins are cytoplasmic and their presence in the cell wall is due to contamination, with the notable exception of tyrosinase (SCAB85681) which is a known Tat substrate that lacks a signal peptide and is transported through the Tat system by forming a complex with a signal peptide-bearing partner protein (Chen et al., 1992; Leu et al., 1992; Schaerlaekens et al., 2001). Verification of S. scabies Tat-targeting signal peptides using the agarase reporter assay Using a combination of bioinformatic and proteomic approaches described above we identified a large number of candidate Tat substrates in S. scabies. A common method for ascertaining whether a given protein is likely to be a Tat substrate is to fuse its signal peptide to a reporter protein and determine whether it is able to mediate export by the Tat pathway. Recently, Widdick et al. (2006; 2008) described the agarase reporter system as a convenient and reliable reporter to test for Tat-dependent export. Agarase is a Tat-exported extracellular enzyme produced by S. coelicolor whose extracellular activity can be readily detected by the formation of halos on agar plates after staining with iodine. Agarase cannot be exported in an active form by the Sec pathway, but has been shown to be exported by the Streptomyces Tat pathway when fused to Tat signal peptide sequences from a wide range of organisms including Gram negative bacteria, archaea and even eukaryotes (Widdick et al., 2006; 2008). Because the size of the halo is related to the amount of agarase secreted, the assay is also semi-quantitative. We therefore selected 35 proteins that had been identified from the extracellular proteome of the WT strain, including 13 from Table 1 that were predicted to be Tat substrates by both TATFIND 1.4 and TatP, three identified by TATFIND 1.4 only, two identified by TatP only, a further 16 that had recognizable signal peptides that were not predicted to be Tat-targeting by either prediction programme, and one that as presented had no apparent signal peptide but if an upstream start site was used it had a good candidate Tat signal peptide. The signal peptides from each of these proteins were fused in-frame to the mature region of agarase. The constructs are designed such that each clone carries the dagA ribosome binding site, with identical spacing between the ribosome-binding site and the start codon (Widdick et al., 2006; 2008). The recombinant proteins were expressed in both S. lividans WT and DtatC strains, and scored for agarase activity. In total, 14 of the 35 signal peptides were able to mediate Tat-dependent export of agarase (Fig. 3A) and these proteins are listed in Table 2. Of the proteins that had signal peptides that could export agarase, ten of them were predicted to have Tat signal peptides by both programmes, and a further two by one or other programme. The remaining two (SCAB15581 and SCAB68191) did not have Tat signal peptides on the ORFs as called, but if plausible upstream start codons were used both had very good signal peptides that were recognized as Tat-targeting by both TATFIND 1.4 and TatP. This observation strongly suggests that the start codons of these two proteins have been mis-annotated (http://www.sanger.ac.uk/Projects/S_scabies/). Interestingly, for two of the signal peptides that were able to mediate export of agarase, no agarase activity was detected if alternative versions of these signal peptides were tested. For SCAB15581, if the n-region of the signal peptide was truncated by selecting an alternative start codon closer to the twin arginine motif, no agarase © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 258 M. V. Joshi et al. 䊏 Table 1. Proteins that are predominantly found in the extracellular fraction of the S. scabies wild-type strain following 2D gel analysis. Protein Growth medium detected Pass TATFIND 1.4 and TatP SCAB08951 R5 SCAB15571 IPM, OBM, R5 SCAB16551 OBM, SFM SCAB16651 IPM SCAB25591 IPM, OBM, R5, SFM SCAB38731 OBM SCAB47131 IPM, OBM SCAB59671 R5 SCAB63891 IPM, SFM SCAB68841 IPM, OBM, R5, SFM SCAB74351 IPM, OBM, R5, SFM SCAB75721 IPM, OBM, R5, SFM SCAB77971 IPM, OBM, R5, SFM SCAB81841 OBM Pass TATFIND 1.4 only SCAB18501 IPM, OBM, R5, SFM SCAB27931 SFM SCAB37611 IPM, OBM, R5, SFM OBM, R5 SCAB57891c SCAB82451 SFM Pass TatP only SCAB08221 IPM, OBM, R5, SFM SCAB64081 IPM, SFM SCAB78761 OBM SCAB84861 R5 Pass Signal P but not TATFIND 1.4 or TatP SCAB01191 IPM SCAB04761 R5 SCAB05351 R5 SCAB07651 IPM SCAB08871 IPM, OBM SCAB13321 R5 SCAB13551 SFM SCAB14911 SFM SCAB16431 OBM SCAB16711 IPM, OBM, R5, SFM SCAB16721 IPM, OBM, R5, SFM SCAB17001 IPM, SFM SCAB17571 IPM SCAB18081 IPM, SFM SCAB18661 SFM SCAB19481 IPM SCAB19491 SFM SCAB19941 SFM SCAB24731 OBM SCAB24891 SFM SCAB26841 SFM SCAB27771 OBM SCAB31531 IPM, R5 SCAB33981 SCAB36371 SCAB41181 SCAB43901 SCAB44001 SCAB44161 SCAB44541 SCAB44691 SCAB45141 SCAB47841 SCAB49491 SCAB56441 SCAB58251 SCAB59651 SCAB66031 SCAB68191c SFM IPM SFM IPM SFM R5, SFM IPM, OBM IPM, OBM, SFM SFM R5, SFM IPM, OBM, R5, SFM IPM SFM SFM SFM IPM, OBM, R5, SFM Putative function Agarase testa ABC-type Fe3+ transport system, periplasmic component Putative secreted phosphoesterase Putative mannosidase Putative secreted protein Putative secreted cellulase Putative secreted beta-lactamase Peptidase family M23/M37 protein Putative hydrolase Putative secreted transport-associated protein Putative 5′ nucleotidase Glycerophosphoryl diester phosphodiesterase Putative secreted protein Putative secreted phosphatase (fragment) Putative secreted hydrolase Pass Pass ND Pass Pass Pass Failb Pass Failb Pass Pass Pass Failb Pass Alpha-N-acetylglucosaminidase Putative neutral zinc metalloprotease Putative secreted aminopeptidase Conserved hypothetical protein Putative secreted protein Pass Failb Fail ND ND Conserved hypothetical protein Putative secreted protein Putative secreted protein Putative secreted amidase Pass Fail ND ND Putative secreted protein Putative secreted protein Substrate-binding component of ABC transporter Neutral zinc metalloprotease Secreted endoglucanase S15 non-peptidase homologue family Conserved hypothetical protein Putative secreted protein Putative possible cellulase CELA1 Putative secreted glycosyl hydrolase Putative secreted glycosyl hydrolase Secreted cellulase Putative secreted peptidase Gamma-glutamyltranspeptidase Putative serine protease RTX-family exoprotein Putative probable exported protein Cyclase Putative secreted aminopepetidase Glutamate-binding protein Putative serine protease Putative secreted protein Putative BldKB-like transport system extracellular solute-binding protein Hypothetical protein 2SCK36.08 Putative xylanase/cellulase Conserved hypothetical protein Putative secreted hydrolase Putative secreted protein Hydrolase Putative secreted protein Putative secreted protein D-alanyl-D-alanine carboxypeptidase Lipoprotein Putative secreted 5′-nucleotidase Putative secreted protease Putative secreted tripeptidyl aminopeptidase Putative secreted serine protease Putative glycosyl hydrolase Putative secreted alkaline phosphatase ND ND Fail Fail ND ND Fail ND ND Failb Fail ND Fail ND ND ND ND ND ND ND ND ND ND Fail ND Fail ND ND ND Fail ND ND ND ND ND Fail ND Passb © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 The Tat pathway in S. scabies 259 Table 1. cont. Protein Growth medium detected Putative function Agarase testa SCAB68931 SCAB69691 SCAB72441 SCAB72781 SCAB74081 SCAB78431 SCAB80071 SCAB84971c SCAB85291 SCAB90091 SCAB90101 No signal peptide SCAB15581c SCAB17551 SCAB20121 SCAB21191 SCAB25251 SCAB26011 SCAB31831 SCAB36931 SCAB37201 SCAB39491 SCAB39811 SCAB41891 SCAB44821c SCAB45751 SCAB50441 SCAB51341 SCAB51541 SCAB54151 SCAB55881 SCAB57721 SCAB58791 SCAB59311 SCAB59871 SCAB62141 SCAB64141 SCAB64311 SCAB67061 SCAB69391 SCAB71391 SCAB85681d SFM IPM IPM, SFM IPM IPM, OBM, R5 SFM SFM IPM IPM IPM, OBM, SFM SFM Branched chain amino acid-binding protein Zinc-binding carboxypeptidase Putative membrane protein Penicillin acylase Putative secreted protein Secreted tripeptidylaminopeptidase Putative secreted protein Conserved hypothetical protein Conserved hypothetical protein Secreted cellulase Secreted cellulase ND Fail Failb ND ND Fail ND Fail ND fail ND IPM, OBM, R5, SFM IPM R5, SFM IPM, SFM OBM OBM R5 R5 OBM OBM, R5, SFM R5 R5 IPM, OBM, R5, SFM R5 R5 OBM, R5 SFM OBM IPM OBM R5 R5 SFM SFM SFM SFM SFM R5 OBM R5 Conserved hypothetical protein Conserved hypothetical protein Putative germacradienol synthase Hypothetical protein Guanosine pentaphosphate synthetase Elongation factor Ts Cytochrome P-450 hydroxylase 50S ribosomal protein L4 50S ribosomal protein L7/L12 Clp-family ATP-binding protease Conserved hypothetical protein Adenylosuccinate synthetase Conserved hypothetical protein DNA gyrase subunit A Chaperonin 2 Citrate synthase Calcium binding protein Ribosomal L25p family protein Transcriptional regulator Cellobiose hydrolase Citrate synthase ABC transporter ATP-binding protein GntR family DNA-binding regulator Pyruvate phosphate dikinase Acyl carrier protein Conserved hypothetical protein Dihydrolipoamide dehydrogenase Conserved hypothetical protein Conserved hypothetical protein Putative tyrosinase MelC2 Passb ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND a. Signal peptide sequences (listed in Table S5) of the indicated proteins were fused to the mature region of agarase and their ability to export agarase in a Tat-dependent manner was determined as described in the text. b. Two variants of each of these signal peptides were tested (see Table S5). c. The N-termini of these proteins are recognized as Tat signal peptides by both TATFIND 1.4 and TatP if alternative start sites are selected. d. This protein lacks an N-terminal signal peptide but is a Tat substrate by virtue of interacting with a Tat signal peptide-bearing partner protein, MelC1 (Leu et al., 1992; Schaerlaekens et al., 2001). ND, not determined. activity was detected. For SCAB68191, which does not have a twin arginine motif in the ORF as called, two alternative start codons were selected that both produced peptides containing a twin arginine motif but with different lengths of n-region. Again only the peptide with the longer n-region gave any detectable agarase activity. This indicates that the choice of start codon may be critical to whether a given signal peptide is able to mediate agarase export. Surprisingly, three of the signal peptides in Table 2 that were predicted to be Tat-targeting by both TATFIND 1.4 and TatP did not mediate detectable export of agarase. The reason for this is not clear; however, given the observation that choice of start site can significantly affect the extracellular agarase activity, it is possible that incorrect start sites were selected for these signal peptides. This may be particularly relevant for SCAB77971, which is a predicted PhoD family phosphatase that are known to have very long and variable signal peptide n-regions (Widdick et al., 2006; 2008). Alternatively, it is possible that these are either not Tat-targeting signals or that they are genuine Tat-targeting signals, but that they do not © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 260 M. V. Joshi et al. 䊏 Fig. 3. Export of agarase mediated by S. scabies signal peptides. The x-axis shows a range of signal peptides from S. scabies from either (A) cell wall-associated proteins that were differentially absent from the DtatC strain (selected from the list in Table 2) or (B) that were predicted to be Tat signal peptides by bioinformatic analysis (listed in Table S1). The y-axis gives a measure of agarase export from each fusion protein, compared with agarase bearing its native signal peptide (DagA) cloned in the same manner (set at 100%). Each construct carries the native agarase promoter and ribosome binding site. The error bars represent the standard error of the mean, where n = 3. A. Signal peptides from the following proteins listed in Table 1 were also tested and found to be negative in this assay: SCAB07651, SCAB13551, SCAB16711 (two variants), SCAB16721, SCAB17571, SCAB27931 (two variants), SCAB36371, SCAB37611, SCAB43901, SCAB44691, SCAB47131 (two variants), SCAB59651, SCAB63891 (two variants), SCAB64081, SCAB69691, SCAB72441 (two variants), SCAB77971 (two variants), SCAB78431, SCAB84971, SCAB90091. B. Two variants of the SCAB63071 signal peptide were able to mediate export of agarase to differing degrees – both are shown in the Figure. Signal peptides from the following proteins listed in Table S1 were also tested and found to be negative in this assay: SCAB08301, SCAB31461, SCAB34181, SCAB70581 and SCAB78851. The exact amino acid sequences of each of the sequences tested in these assays can be found in Table S5. direct the Tat-dependent export of agarase for some unknown reason. In addition to testing signal peptides from proteins shown in the proteomic study to be predominantly present in the extracellular protein fraction of the WT strain, we also tested a further 38 signal peptides that were predicted to be Tat-targeting based on the bioinformatic analysis of the S. scabies genome sequence described above (and listed in Table S1). These 38 proteins are made up of 28 proteins that were predicted to have Tat signal peptides based on the fact that they were recog- nized by TATFIND 1.4 and TatP, six that were recognized by TATFIND 1.4 and TatP following reassignment of the start codon, two that were recognized by both prediction programmes after in silico truncation of the N-terminal part of the ORF, one protein that was predicted to have a Tat signal peptide by TATFIND 1.4 only, and one that was predicted by TatP only. As shown in Fig. 3B, signal peptides derived from 33 of the proteins tested were able to mediate export of agarase and we therefore conclude that these proteins are Tat substrates. The proteins that bear these signals © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 The Tat pathway in S. scabies 261 Table 2. Proteins from S. scabies with signal peptides that are able to mediate the Tat-dependent export of agarase. Signal peptide tested Putative function Protein detected by proteomics SCAB08221 SCAB08951 SCAB15571 SCAB15581 SCAB16651 SCAB18501 SCAB25591 SCAB38731 SCAB59671 SCAB68191 SCAB68841 SCAB74351 SCAB75721 SCAB81841 Proteins predicted by bioinformatics SCAB00601 SCAB03871 SCAB04961 SCAB06471 SCAB09381 SCAB09591 SCAB10131 SCAB13731 SCAB15711 SCAB16951 SCAB19551 SCAB25621 SCAB37061 SCAB46711 SCAB48941 SCAB57331 SCAB57661 SCAB63071 SCAB66251 SCAB73161 SCAB74641 SCAB74681 SCAB77391 SCAB77401 SCAB77511 SCAB78741 SCAB79011 SCAB79571 SCAB80581 SCAB81041 SCAB81901 SCAB88501 SCAB89661 Putative phosphoesterase ABC-type Fe3+ transport system, periplasmic component Putative secreted phosphoesterase Possible histidinol phosphatase (PHP family) Putative L-xylulose-5-phosphate 3-epimerase Alpha-N-acetyl glucosaminidase Endo-1,4-beta-glucanase Beta-lactamase Cyclic 3′,5′-adenosine monophosphate phosphodiesterase Phospholipase D precursor 2′,3′-cyclic-nucleotide 2′-phosphodiesterase Glycerophosphoryl diester phosphodiesterase A subfamily of peptidase family C39 Alpha-L-rhamnosidase Hypothetical protein Glycosyl hydrolase domain followed by ricin domain Endo-xylanase Putative alpha-L-fucosidase Hypothetical protein Alpha-L-fucosidase Glycosyl hydrolase domain followed by ricin domain Phospholipase C Putative endo-1,3-beta-glucanase Hypothetical protein Rhamnogalacturonase B precursor Lipoprotein Putative secreted protein Protocatechuate dioxygenase ATP-dependent nuclease subunit B-like No homologues in database Putative multiple sugar ABC transporter solute-binding protein Putative gluconolactonase Iron-dependent peroxidase Gluconolactonase Lipoprotein Hypothetical protein Hypothetical Protein Glycosyl hydrolase Beta-galactosidase Putative secreted protein Lipase/acylhydrolase, putative Large secreted protein Amine oxidase Putative ABC transporter periplasmic binding protein Peptide ABC transporter peptide-binding protein Rhamnogalacturonase B precursor Putative factor C protein precursor The amino sequences of each signal peptide tested are given in Table S5. are also listed in Table 2. Of the signal peptides tested in this section, two variants of the SCAB00601 signal peptide were tested, of which only the clone that encoded a signal peptide with the longer n-region (pSM29b, see Table S5), gave any detectable activity. Likewise, three variants of the SCAB63071 signal peptide were tested. Of these, the agarase-producing clone that encoded the longest n-region (pSM35a) failed to give detectable agarase activity while the remaining two both gave detectable agarase export but at different levels. This indicates that the features of the cloned DNA sequence and/or the sequence of the signal peptide tested in this assay may have a profound effect on the outcome of the reporter assay. Five of the signal peptides identified as Tat-targeting by bioinformatic means (SCAB08301, SCAB31461, SCAB34181, SCAB70581 and SCAB78851) did not promote the export of agarase. Again the reasons for this is not clear – it may be related to the fact that inappropriate start codons were selected for these signal peptides, © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 262 M. V. Joshi et al. 䊏 Fig. 4. Reduced disease severity of Arabidopsis seedlings inoculated with the DtatC mutant strain. Arabidopsis seedlings (7 days old) were inoculated with spores (corresponding to 1 ¥ 106 cfu) of the S. scabies WT (87.22) and DtatC mutant (DtatC) strains, keeping un-inoculated seedlings as controls. The plants were grown for a further 21–28 days, and the images are taken at 28 days post inoculation. or that they were genuine Tat signal peptides but were unable to mediate detectable export of agarase. Alternatively, it is possible that despite the fact they were predicted to be Tat signal peptides by both TATFIND and TatP they are actually Sec-targeting. In this context it is interesting to note that SCAB31461 is the S. scabies homologue of S. coelicolor BldKB that has a KR rather than an RR motif in its signal peptide and is also unable to mediate export of agarase (Widdick et al., 2006). This would be consistent with the idea that this protein may be a Sec substrate in Streptomyces. In total, the agarase reporter assay has identified 47 Tat-targeting sequences, four of which were predicted to be Tat-targeting by only one of the two prediction programmes, and we conclude that it is highly likely that these proteins are Tat substrates in S. scabies. The Tat secretion system contributes to the virulence of S. scabies The demonstrated role of Tat-secreted proteins in the virulence of other microbial pathogens (reviewed in De Buck et al., 2008) and the existence of proteins in the S. scabies genome that have homologues in pathogenic microbes, suggested a likely role for Tat secretion in virulence. We therefore investigated the virulence of the DtatC mutant, relative to the WT strain, on the model plant Arabidopsis. S. scabies is a broad host range pathogen that causes root rot on both model plant species and agricultural crops (reviewed in Loria et al., 2006). The root tips of Arabidopsis (Col-O) seedlings were inoculated with S. scabies spores from either the WT (WT; 87-22) or the DtatC mutant, and seedling growth was monitored weekly. As shown in Fig. 4, infection of seedlings by the WT strain caused root stunting and necrosis; secondary roots were killed soon after emergence from the taproot. In addition, the leaves and shoots were stunted, chlorotic and necrotic; all of the plants inoculated with the WT strain were dead within 21–30 days post inoculation. By contrast, plants inoculated with the DtatC mutant strain grew vigorously and were similar to the un-inoculated control in size and colour (Fig. 4). Interestingly, the DtatC mutant grew extensively on the roots as yellow substrate mycelium, without noticeable sporulation, but did not appear to necrotize the colonized tissue. Plants inoculated with the DtatC mutant did, however, differ from the un-inoculated control in root morphology, particularly root-branching pattern (Fig. 4). Given the dramatic virulence phenotype of the DtatC mutant, we carried out a time course study of Arabidopsis root colonization using confocal scanning microscopy and enhanced green fluorescent protein (EGFP)-labelled S. scabies WT and DtatC strains. To facilitate observation of microbial growth, plants were grown hydroponically. The WT strain attached to the root tip and began to colonize plant tissues within 24 h after spores were added to the hydroponic medium (Fig. 5A). Within 48 hpi the WT strain was aggressively colonizing the root at the zone of cell differentiation (Fig. 5B) and at lateral meristems (Fig. 5C). Intercellular colonization was evident at the root tip and at the point of secondary root emergence by 72 hpi (Fig. 5D). By contrast, colonization of root tissue by the DtatC mutant was greatly delayed and limited in scope. The mutant was not able to attach to the root tip during the first 48 hpi; however, there were loosely attached colonies at the root elongation zone (Fig. 5E), and minimal colonization and restricted growth at the differentiation and elongation zones (Fig. 5F–G), and at lateral meristems (Fig. 5H) after 72–96 hpi. Intercellular colonization was delayed until 96–120 hpi (Fig. 5I–J) and was limited in scope. Individual Tat-secreted proteins contribute to the virulence of S. scabies Because the DtatC strain was essentially avirulent on Arabidopsis seedlings, we sought to address the contribution of individual candidate Tat substrates to host– pathogen interactions. Fourteen individual strains were constructed that were deleted for genes encoding the putative or confirmed Tat substrate proteins listed in Table 3. The strains were then assessed for virulence using Arabidopsis seedlings as a host. To this end, Arabidopsis seedlings were grown on Murashige and Skoog © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 The Tat pathway in S. scabies 263 Fig. 5. Micrographs of Arabidopsis roots colonized by EGFP-labelled S. scabies strains. Colonization and disease progression was monitored on hydroponically grown Arabidopsis roots after inoculation with EGFP labelled S. scabies strains. (A–D) Inoculation with the WT strain 87-22, and (E–J) inoculation with the DtatC mutant strain. Colonies of the WT strain are shown (A) 24 h post inoculation (hpi) showing cells at and near the root tip (red arrow); (B and C) 48 hpi showing colonization of (B) the root at the zone of cell differentiation and (C) at a lateral meristem; and (D) 72 hpi at a lateral root meristem – intercellular growth is indicated by the red arrows. Colonies of the tatC mutant strain are shown (E) at the root tip zone during the first 24–48 hpi; the arrow indicates loosely attached mycelium at the elongation zone; (F–H) after 72–96 hpi; (F) at the differentiation zone (G) at elongation zones and (H) at a lateral meristem; and (I and J) at 96–120 hpi. (K and L) Un-inoculated control seedlings showing (K) normal root growth, and (L) loose cells surrounding the root cap region. Images were taken using a Leica TCS SP5 confocal microscope. Size bars represent 50 mm. (MS) medium and inoculated with spore suspensions of the WT or with mutant strains containing deletions in genes encoding candidate Tat substrates using methods just described. Of the 14 individual mutant strains tested, seven were reduced in virulence relative to the WT strain (Fig. 6). Gene deletions in SCAB03871 and SCAB10131 were among those coding for Tat substrates that have a reduced virulence phenotype in the Arabidopsis seedling assay. Interestingly, these genes encode paralogues with predicted glycosyl hydrolase and ricin-like domains. In spite of the similarity in their amino acid sequences, each of these proteins appears to make a large and unique contribution to virulence, based on the phenotypes of the individual mutants (Fig. 6). Furthermore, proteins encoded by SCAB03871 and SCAB10131 have homologues in fungal plant pathogens such as Chaetomium globosum (CHGG_02005) and Phaeosphaeria nodorum (SNOG_01152) respectively. SCAB06471 encodes a putative alpha-L-fucosidase; the cognate deletion mutant was compromised in virulence (Fig. 6). Alpha-L-fucosidases are responsible for processing of fucosylated glycoconjugates that play a role in a wide variety of biological processes. Interestingly, © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 264 M. V. Joshi et al. 䊏 Table 3. Potential Tat-dependent virulence factors encoded in the genome of S. scabies. Gene ID Putative function SCAB03871 Glycosyl hydrolase domain followed by ricin domain SCAB06471 Putative alpha-L-fucosidase SCAB08951 ABC-type Fe3+ transport system, periplasmic component Glycosyl hydrolase domain followed by ricin domain SCAB10131 SCAB18791 Hypothetical secreted protein SCAB58511 Hypothetical protein. SCAB58531 Hypothetical protein SCAB70581 Hydrolase of the alpha/beta superfamily SCAB76661 Rare lipoprotein A SCAB77391 Conserved hypothetical protein containing a galactose-binding domain Putative glycosyl hydrolase SCAB77401 SCAB80581 Putative FAD-containing amine oxidase SCAB81041 Putative spermidine/putrescine transporter peptide-binding protein Alpha-L-rhamnosidase SCAB81841 Homologs with highest similarity (identity/similarity) Locus_tag Streptomyces sviceus (89/93) Micromonospora aurantiaca (76/85) Actinosynnema mirum (71/81) Streptomyces sviceus (82/90) Streptomyces viridochromogenes(83/89) Streptomyces hygroscopicus (57/70) Acidovorax delafieldii (60/74) Phytophthora infestans (51/67) Pectobacterium carotovorum (48/63) Streptomyces sviceus (84/92) Micromonospora sp. L5 (75/83) Actinosynnema mirum (70/80) Bacteroides cellulosilyticus (39/57) Pectobacterium carotovorum (34/50) Ralstonia solanacearum (34/49) Pseudomonas putida (27/45) Nitrobacter winogradskyi (27/43) Clostridium botulinum A3 (25/42) Nitrobacter winogradskyi (29/45) Clostridium botulinum Ba4 (26/43) Pseudomonas putida (29/45) Streptomyces sviceus (76/82) Streptomyces griseoflavus (76/83) Streptomyces ghanaensis (76/83) Streptomyces sviceus (54/65) Micromonospora sp. (66/79) Streptomyces viridochromogenes (72/82) Bacteroides cellulosilyticus (36/53) Pectobacterium carotovorum (30/47) Ralstonia solanacearum (29/44) Bacteroides ovatus (45/63) Dickeya dadantii (36/52) Ralstonia solanacearum (36/52) Streptomyces pristinaespiralis (78/87) Streptomyces sp. (76/85) Nocardia farcinica (54/65) Streptomyces sviceus (58/72) SSEG_02060 MicauDRAFT_3913 Amir_3107 SSEG_02413 SvirD4_010100003282 ShygA5_010100010179 AcdelDRAFT_2816 PITG_03258 PC1_1331 SSEG_02060 ML5DRAFT_2741 Amir_3107 BACCELL_04049 PC1_0414 RSIPO_04319 PP_2006 Nwi_0886 CLK_A0070 Nwi_0886 CLJ_0010 PP_2006 SSEG_08785 SgriT_010100028100 SghaA1_010100027639 SSEG_04607 MCAG_02629 SvirD4_010100009886 BACCELL_04049 PC1_0414 RRSL_03557 BACOVA_01686 Dd586_1768 RSMK02989 SSDG_02025 StreC_010100030349 nfa3040 SSEG_09980 Streptomyces hygroscopicus(56/72) ShygA5_010100044540 Sphaerobacter thermophilus (36/53) Sthe_3297 Clostridium leptum (35/54) Victivallis vadensis (37/51) Lactobacillus rhamnosus (33/48) CLOLEP_02977 Vvad_PD1953 LRH_00532 Agarase testa Virulence phenotypeb Yes Yes Yes Yes Yes No Yes Yes ND No ND No ND No ND No ND No Yes Yes Yes No Yes Yes Yes Yes Yes Yes a. Signal peptide sequences (listed in Table S5) of the indicated proteins were fused to the mature region of agarase and their ability to export agarase in a Tat-dependent manner was determined as described in the text. b. Virulence phenotype of knock-out strains tested using the Arabidposis thaliana infection model. ND, not determined. SCAB06471 has homologues in the fungal plant pathogens such as Gibberella zeae (FG11254.1) and Magnaporthe grisea (MGG_03257). Furthermore, all three of these proteins are putative glycosidases and the cognate mutants have similar virulence phenotypes; in all cases plant growth was substantially greater than those inoculated with the WT strain, root necrosis was lacking, and the mutant strains grew luxuriously on Arabidopsis roots (Fig. 6). It is tempting to speculate that these proteins have a role in penetration of the plant cell wall. The SCAB77391 mutant strain was slightly reduced in virulence, relative to WT (Fig. 6). The encoded protein is predicted to contain a galactose-binding domain. It is possible that this domain recognizes specific carbohydrate moieties on the host cell surface; in that way the protein might function as a lectin, which could enhance host binding or recognition. The limited virulence phenotype, however, suggests a function that is either redundant in the genome or has only a minor role in host–pathogen interactions. The SCAB81841 mutant strain has a dramatic avirulence phenotype; plants inoculated with this mutant were comparable to the non-inoculated control, except for leaf chlorosis and a delay in flowering. The predicted function © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 The Tat pathway in S. scabies 265 Fig. 6. Tat substrates are essential for the virulence of S. scabies. Deletion mutants of putative Tat-secreted virulence proteins are constructed using the ReDirect gene disruption protocol and compared to WT 87-22 for disease severity. Mutants SCAB77391, SCAB80581, SCAB81041, SCAB81841, SCAB03871, SCAB06471 and SCAB10131 show suppression of disease severity in terms of root necrosis and aerial growth. for the encoded protein is an alpha-L-rhamnosidase, which hydrolyses the terminal non-reducing alpha-Lrhamnose residues in alpha-L-rhamnosides. Given the severe defect in virulence, it seems unlikely that this enzyme’s primary function is to hydrolyse plant biomass. Some rhamnosides are biologically active, including antimicrobial saponins (Morrissey et al., 2000). Interestingly, the SCAB81841 mutant grew poorly on Arabidopsis roots (Fig. 6), consistent with a role in the degradation of an antimicrobial molecule. The SCAB81041 deletion mutant showed a moderate virulence phenotype. The cognate protein encodes a putative spermidine/putrescine transporter peptidebinding protein (Table 3). SCAB80581 encodes a putative FAD-containing amine oxidase. Proteins in this family additionally contain a second domain that is responsible for specifically binding a substrate and catalysing a particular enzymatic reaction. The deletion mutant has a moderate phenotype as shown in Fig. 6. Discussion In this study we have characterized the Tat secretome of the plant pathogen, S. scabies. In keeping with analyses of other streptomycete genomes, in silico analysis using Tat substrate prediction programmes TATFIND 1.4 and TatP predicts many candidate Tat substrates are encoded by S. scabies. Using the agarase reporter assay, we show here that the signal peptides of 47 candidate S. scabies Tat substrates were able to mediate Tat-dependent export, strongly suggesting that these represent bona fide Tat substrates. Interestingly, two of the Tat signal peptides identified in this study were not recognized by TATFIND 1.4 because they have hydrophobic amino acids in the +1 position of the twin arginine motif. Stanley et al. (2000) previously noted that the amino acid residue at this position is usually polar, but the work presented here shows that valine and leucine can both be tolerated at this position. Furthermore, additional highly likely Tatdependent proteins listed in Tables S1 and S2 also have signal peptides that are not recognized by TATFIND 1.4 due to the presence of His, Ile or Phe at the +1 position. Further work is required to ascertain whether these additional ‘allowed’ residues at the +1 position are specific to Streptomyces proteins, or whether they reflect a general tolerance for the nature of the amino acid at this position of Tat signal peptides. None-the-less, taken together our analysis supports the contention that there are well in excess of 100 Tat substrates in S. scabies. Consistent with the notion that the S. scabies Tat pathway is a major route of protein secretion, inactivation © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 266 M. V. Joshi et al. 䊏 of the Tat pathway was associated with a number of phenotypes. In particular, growth rate of the DtatC strain was reduced relative to WT and the mutant displayed a cell wall defect, a phenotype that is also linked to Tat pathway inactivation in other bacteria (e.g. Ize et al., 2003; Caldelari et al., 2006; Widdick et al., 2006). The DtatC strain was also slow to sporulate on some types of solid media. Interestingly, the signal peptide of the S. scabies homologue of the extracellular signalling protein factor C (SCAB89661), that is involved in cellular differentiation in some species of Streptomyces (Birkó et al., 1999) was shown to mediate Tat-dependent export of agarase (Fig. 3B). This indicates that the S. scabies factor C homologue is a Tat substrate. The twin arginine signal peptides of streptomycete factor C proteins are highly conserved and the mature portion of the protein shows strong sequence identity to secreted proteins encoded by mycelial fungi, raising the possibility that this DNA coding this protein was acquired by lateral gene transfer (Chater et al., 2010). Analysis of the extracellular proteomes of the WT and DtatC strains showed that in total 73 predicted secreted proteins were present at decreased amounts or absent in the extracellular proteome of the DtatC mutant. Using the agarase reporter assay, 14 predicted Tat substrates detected by proteomics as being abundantly exported in more than one growth condition in WT cells and strongly reduced in the DtatC mutant were verified to have Tattargeting signal peptides. However, the majority of proteins that were apparently absent from the DtatC cell wall wash fraction are unlikely to be Tat substrates, again underscoring the pleiotropic nature of the mutation. Bacteria in symbiotic associations with eukaryotes rely on secreted molecules to manipulate host cell physiology. In the broadest definition, the small molecules and proteins that alter host cell structure and function are referred to as ‘effectors’ (Hogenhout et al., 2009). In S. scabies thaxtomin and a coronatine-like molecule meet the criteria of small molecule effectors. Thaxtomin acts as a cellulose synthesis inhibitor, through an undefined mechanism, and is believed to aid in penetration of plant tissue (Scheible et al., 2003; Bischoff et al., 2009). A coronatine-like molecule likely serves as a molecular mimic of the plant defence signalling molecule, jasmonic acid (Bignell et al., 2010). The secreted proteins Nec1 and TomA are authentic and putative effectors, respectively, in S. scabies but other effector proteins had not previously been identified. Effector proteins interact with host cells in many ways and the host cell targets of most effectors are not known (Cunnac et al., 2009); however, molecular mimicry and modification of host cell molecules are two broad categories of virulence mechanisms. Activity, abundance and localization of eukaryotic proteins, including those involved in host defence, can be regulated through attach- ment of ubiquitin or ubiquitin-like molecules. Many pathogens produce proteins that mimic components of the host ubiquitination machinery (reviewed in Spallek et al., 2009). Other defence and development signalling cascades are targeted by effector proteins, such as Rho GTPase regulators, cytoskeletal modulators and host innate immunity (Shames et al., 2009). Under the broad definition of protein effectors suggested by Hogenhout et al. (2009), secreted enzymes involved in degradation of host cell molecules, particularly plant cell wall components, are also considered to be effector proteins. Microbial pathogens can undergo rapid evolution through the acquisition of pathogenicity islands (PAIs), which are regions in the genome encoding multiple virulence-associated genes (Lovell et al., 2009). In Gram negative pathogens, PAIs often have a modular configuration and include genes encoding both the type III secretion apparatus and effector proteins that use that pathway (Lindeberg et al., 2009). The type III secretion apparatus provides a very effective mechanism for introduction of effector proteins directly into host cells, but is lacking in Gram positive bacteria. Gram positive pathogens, such as S. scabies, have PAIs (Kers et al., 2005) but must rely on general protein secretion systems, such as Tat and Sec, which deliver effector proteins outside of the bacterial cell; genes encoding effector proteins are not typically clustered with those encoding protein secretion machineries in these pathogens. Translocation of putative virulence factors is Tatdependent in many microbial pathogens including those that contain type III secretion systems (reviewed in De Buck et al., 2008). Given the importance of the Tat system in the genus Streptomyces, it was not surprising to find that inactivation of the Tat secretion machinery in S. scabies resulted in an essentially avirulent phenotype (Figs 4 and 5). The tatC mutant strain was slow to colonize and invade rapidly expanding root tissue, relative to the WT strain, which likely was the result of several deficiencies. However the avirulence phenotype of the S. scabies tatC mutant strain was not due to a deficiency in thaxtomin production (Fig. S1). Regardless, the dramatic virulence phenotype of the DtatC mutant did suggest that a subset of Tat substrates has virulence functions. Furthermore, the combination of bioinformatic and proteomic analysis identified a large number of putative Tat substrates in S. scabies, of which more than one-third are associated with stress responses or virulence in other bacterial or fungal pathogens. This group of putative Tat-secreted virulence proteins includes putative lipoproteins, ABC transporters, phospholipases/ phosphoesterases, beta-lactamase and proteins involved in Fe homeostasis (see Tables 3 and S1). To confirm that Tat substrates are involved in infection, we inactivated genes encoding 14 candidate Tat substrates, from which strains inactivated for production of © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 The Tat pathway in S. scabies 267 seven confirmed Tat substrates showed a reduction in virulence (Fig. 6). Because the growth rate of these mutant strains was comparable to the WT (shown in Fig. S4 for the SCAB03871, SCAB06471, SCAB10131 and SCAB77391 knockout strains), it can be concluded that the Tat pathway secretes multiple virulence factors in S. scabies. Inspection of the list of these authentic virulence proteins using bioinformatics reveals some interesting ecological and phylogenetic associations. Out of the seven Tatsecreted proteins affecting virulence, SCAB77391 and SCAB81841 are conserved in S. turgidiscabies, but the encoding genes are absent from the genomes of nonpathogenic streptomycetes for which genome sequence data are available at this time (data not presented). SCAB03871, SCAB06471 and SCAB10131 all contain homologues in fungal plant pathogens and are all predicted to interact with glycans (Table 3). Surprisingly, the amino acid sequences of the Tat secretion signals of these three proteins are highly conserved, possibly suggesting a common origin. SCAB03871 and its paralogue (confirmed by reciprocal BLAST analysis) SCAB10131 have glycosyl hydrolase and ricin-like domains. The closest homologues to these proteins are a few closely related saprophytic actinomycetes, where the twin arginine signal peptide is always conserved. Outside of the actinomycetes, the closest homologues of these proteins are encoded by saprophytic and pathogenic fungi. In keeping with the fact that fungi generally lack the Tat system, the fungal homologues have typical eukaryotic signal peptides, rather than the longer and less hydrophobic Tat signal peptides. This is suggestive that the gene for one of these proteins was acquired by an ancestoral actinomycete through horizontal transfer from a fungus and that a Tat-targeting sequence was acquired to allow secretion of the protein in the prokaryote. It is, however, not readily apparent why these proteins should be substrates for the Tat pathway rather than the Sec system – for example none of them are predicted to bind redox cofactors that would necessitate export in a folded conformation. Substrates for the Sec machinery are transported in an unfolded form and usually interact with cytoplasmic chaperones to maintain them in an unfolded, export competent state. It is possible that proteins coded by genes acquired by horizontal transfer are not recognized by host chaperones as they are nonnative, and it therefore may be advantageous to export such proteins in a folded form. Of the seven Tat-secreted proteins affecting virulence, SCAB06471 is homologous to an alpha-L-fucosidase from G. zeae, the cause of wheat head blight and M. oryzae, the rice blast pathogen (Oh et al., 2008). This enzyme hydrolyses the alpha-1,6-linked fucose joined to the reducing-end N-acetylglucosamine of carbohydrate moieties in glycoproteins. SCAB80581 encodes a protein for amine oxidase, both are homologous to proteins in the nonpathogenic Streptomyces pristinaespiralis and pathogenic Mycobacterium spp. SCAB81041, a putative ABC transporter, is a periplasmic-binding protein and homologous to proteins in Streptomyces sviceus, and in Thermomicrobium roseum, a Gram negative, obligately thermophilic bacterium. Seven putative effector proteins chosen based on bioinformatic analysis did not have a virulence phenotype. One of these was SCAB08951, which was highly similar (Table 3) to proteins encoded by the Gram negative soil bacterium Acidovorax delafieldii, the Gram negative plant pathogen Dickeya dadantii (which both have predicted Sec signal peptides) and the plant pathogenic oomycete Phytophthora infestans (which has a predicted eukaryotic signal peptide). Since effector proteins commonly have a role in host specificity (Lindeberg et al., 2009), evaluation of these mutants on additional host plants, particularly potato, would be necessary. Experimental procedures Strains, plasmids, media and culture conditions Streptomyces scabies strain 87-22 and the cognate DtatC strain (see below) were routinely grown on either IPM (which contained per litre of tap water 50 g SmashR instant potato mash and 12 g agar), International Streptomyces Project medium 2, or International Streptomyces Project medium 4 (BD Biosciences, San Jose, CA) at 28°C. For proteomic analysis, strains were cultured on either IPM, SFM (Hobbs et al., 1989), R5 medium (Thompson et al., 1980) or oat bran broth medium (Goyer et al., 1998). For growth in liquid culture strains were cultured aerobically in TSB (Kieser et al., 2000). Phenotypic growth tests were carried out on DNA medium (Kieser et al., 2000) and halo diameters were determined using the image processing software GIMP (GNU Image Manipulation Program – http://www.gimp.org/). To test candidate signal peptides for Tat dependence in the agarase assay, DNA encoding the signal peptides of interest were cloned in-frame (as NdeI-BamHI/BglII fragments) with the mature agarase sequence in the integrative plasmid pTDW46H (which is identical to pTDW46 except that the apramycin resistance specified by the vector has been replaced by hygromycin resistance from pIJ10700 using the REDIRECT method (Gust et al., 2003) and the oligonucleotide primers hyg_fwd and hyg_rev. The oligonucleotide primer sequences used for signal peptide amplification are listed in Table S4, and the plasmids used in this study are listed in Table S5. Agarase assays performed and quantified as described by Widdick et al. (2006; 2008). Construction and complementation of the S. scabies DtatC strain For construction of the S. scabies DtatC strain, SM1, an approximately 1000 bp region directly upstream of the tatC © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 268 M. V. Joshi et al. 䊏 gene (SCAB73601) was amplified by polymerase chain reaction (PCR) using the oligonucleotides utatcscabf1 and utatcscabr (see Table S4 for a list of oligonucleotide primers used in this study) and S. scabies chromosomal DNA as template, digested with EcoRI and HindIII and cloned into pBluescript (KS+) that had been similarly digested. An approximately 1000 bp region downstream of tatC was subsequently amplified using the oligonucleotides dtatcscabf and dtatcscabr1, digested with EcoRI and XbaI and cloned into pBluescript already containing the tatC upstream region that had been similarly digested. The apramycin resistance cassette from pIJ773 (Gust et al., 2003) was amplified using oligonucleotides ecoriapraf and ecoriaprar, cleaved with EcoRI and cloned between the upstream and downstream regions of the tatC deletion allele assembled in pBluescript. To allow the detection of double-crossover strains by replica plating the ampicillin/carbenicillin resistance cassette of the carrier plasmid was replaced with a kanamycin resistance cassette, which, unlike bla, is suitable for selection in Streptomyces. This was achieved using the REDIRECT method of Gust et al. (2003), and the kanamycin resistance cassette was amplified by PCR using the oligonucleotides KanRtrans2_fwd and KanRtrans2_rev (Table S4). The plasmid was transferred by mating into S. scabies 87-22 and single-crossover recombinants were selected for on MS medium containing apramycin and kanamycin (Gust et al., 2003). Double-crossover recombinants were subsequently selected by several rounds of growth on non-selective media followed by selection for colonies that were apramycin-resistant and kanamycin-sensitive. Loss of the tatC gene in strain SM1 was subsequently confirmed by PCR and by Southern blot analysis. To test for complementation of the S. scabies DtatC strain, a synthetic construct covering the S. scabies tatAC genes (covering from 260 bp upstream of tatA to 60 bp downstream of tatC) in pUC57 was purchased from GenScript, NJ, USA. The tatAC-containing region was excised by digestion with XbaI and cloned into similarly digested pSET-SOR-hyg (Sean O’Rourke, unpublished) to give plasmid pTDW185, which integrates site specifically into the Streptomyces chromosome. Deletion of genes encoding putative Tat substrates Deletion mutants for 14 putative Tat-secreted virulence proteins (Tables 3 and S1) were created using the PCR-based ReDirect gene disruption protocol (Gust et al., 2003). In this case, a cosmid library of S. scabies strain 87-22 containing a kanamycin resistance marker (neo) was used. Cosmid clones containing the target genes were individually introduced into E. coli BW25113 carrying an arabinose-inducible lRed-expressing plasmid pKD46 (AmpR) (Datsenko and Wanner, 2000). A gene replacement cassette [aac(3)IV (ApraR) + oriTRK2] from the plasmid pIJ773 (Gust et al., 2003) was PCR-amplified using gene-specific redirect primers (Table S4). The resulting PCR products were transformed into the cosmid-containing E. coli strain and selected for apramycin resistance. The mutant cosmids were then introduced into S. scabies 87-22 by intergeneric conjugation from E. coli ET12567 (carrying the helper plasmid pUZ8002). Exconjugants were screened for apramycin resistance (100 mg ml-1) and kanamycin sensitivity (50 mg ml-1), indicat- ing a double-crossover allelic exchange in S. scabies; deletions were confirmed by PCR analysis. Protein methods Growth curves for Streptomyces strains were monitored by assaying total protein at regular intervals. To this end, 1 ¥ 106 spores of each strain were inoculated into 100 ml of TSB medium and the cultures were incubated with shaking at 30°C over 56 h. Every 2 h, 3 ¥ 1 ml samples were withdrawn, the cells pelleted and frozen at -20°C. The frozen samples were subsequently resuspended in 1 ml 1N NaOH, 0.1% SDS and boiled for 10 min to lyse the cells. The samples were then clarified by centrifugation and 500 ml of the resulting supernatants were diluted with an equal volume of sterile distilled water, and 25 ml of each sample was used for protein determination using the Biorad DC Protein Assay Kit. For the preparation of extracellular proteins, autoclaved 75 mm cellophane discs were placed onto solid media plates (SFM, OBM, IPM or R5), inoculated with 106 spores of the S. scabies WT and DtatC strains and incubated for 48 h at 30°C. The biomass was scraped from the cellophane discs and extracellular proteins were released following cell wall washing exactly as described by Widdick et al., (2006). Separation of TCA-precipitated cell wall washes of S. scabies extracellular proteins by two-dimensional gel electrophoresis (2D-PAGE) was performed using the immobilized pH gradients in the pH range 3–10 as described (Antelmann et al., 2001). For quantification of the relative protein amounts of extracellular proteins that are decreased in the DtatC mutant proteome compared with the WT, 200 mg protein were separated by 2D-PAGE and the resulting 2D gels were stained with Coomassie-Brilliant Blue as described (Antelmann et al., 2001). Quantitative image analysis was performed from the coomassie-stained 2D gels using the DECODON Delta 2D software (http://www.decodon.com). The 2D gel images from WT and the DtatC mutant cell wall proteomes were aligned using a warp transformation. Before spot detection and quantification was performed, a fused 2D gel of both images was created using the ‘union fusion’ algorithm of Delta2D. Spot detection was performed in the fusion gel containing all spots present in both images according to the automatically suggested parameters for background subtraction, average spot size, and spot sensitivity. The resulting spot shapes were reviewed and manually edited in the fusion gel if necessary. This reviewed spot mask served as a spot detection consensus for all gel images, which was applied to both images to guide the spot detection and quantification. This enables spot quantification in all gels at the same locations resulting in 100% matching and in a reliable analysis of complete expression profiles. Normalization was performed by calculating the quantity of each single spot in percentage related to the total spot quantity per gel. Proteins showing an induction of at least twofold in the WT compared with the DtatC mutant strain are listed in Tables 1, S2 and S3. For standard identification of the proteins from 2D gels, spot cutting, tryptic digestion of the proteins and spotting of the resulting peptides onto the MALDI-targets (Voyager DE-STR, PerSeptive Biosystems) were performed using the Ettan Spot Handling Workstation (Amersham-Biosciences, © 2010 Blackwell Publishing Ltd, Molecular Microbiology, 77, 252–271 The Tat pathway in S. scabies 269 Uppsala, Sweden) as described previously (Eymann et al., 2004). The MALDI-TOF-TOF measurement of spotted peptide solutions was carried out on a Proteome-Analyzer 4800 (Applied Biosystems, Foster City, CA, USA) as described previously (Eymann et al., 2004). The mascot search was performed against the available S. scabies database (http://www.sanger.ac.uk/Projects/S_scabies/). Thaxtomin A extraction and quantification Streptomyces scabies WT 87-22 and the DtatC mutant were grown in 6 ¥ 5 ml of oat bran broth medium (Johnson et al., 2007) in 6-well plates for 7 days at 25 ⫾ 2°C with moderate shaking (~120 r.p.m.). Mycelia were pelleted by centrifugation and discarded. Thaxtomin A was extracted from culture supernatants and was analysed by HPLC as previously described (Johnson et al., 2007). Plant virulence assays Arabidopsis thaliana (ecotype Columbia) surface-sterilized seeds were placed on MS) (Murashige and Skoog, 1962) agar medium with 2% sucrose. Plants were grown at 21 ⫾ 2°C with a 16 h photoperiod for 7 days, then inoculated with S. scabies 87-22 (WT) or the cognate deletion mutants (DtatC mutant or one of the 14 strains harbouring deletions in genes encoding Tat substrates). In all cases, spore suspensions (1x106 cfu) were applied to seedling root tips. Disease symptoms were noted every week and images were taken 21–28 days post inoculation. Each experiment was repeated three times with five replicates of 20–25 plants. Root colonization by the DtatC mutant strain Streptomyces scabies WT 87-22 and the DtatC mutant strains each tagged with a gene-encoding EGFP were created and used for colonization studies with A. thaliana. Vector pIJ8641 (Sun et al., 1999) or pRFSRL16 (R.F. Seipke, unpublished), carrying the egfp gene downstream of the constitutive ermEp* promoter and an antibiotic-resistant marker were replicated in the methylation-deficient E. coli strain ET12567 prior to conjugation into S. scabies WT 87-22 and the DtatC mutant strain respectively. A. thaliana seedlings were grown on liquid MS medium with 2% sucrose for 7 days and inoculated at the root tip with a spore suspension (1 x 106 cfu). Laser scanning confocal microscopy was used to visualize internal and external colonization of Arabidopsis roots at 24 h intervals. The roots of harvested plants were mounted in water immediately after harvesting and observed using a Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany), with a 10 ¥ and 63 ¥ water immersion objectives as described (Joshi et al., 2007a). EGFP was visualized using a 4-line argon laser with an excitation wavelength of 488 nm and an emission wavelength of 500 to 550 nm. Differential interference contrast images were collected simultaneously with the fluorescence images using the transmitted light detector and processed using Leica LAS-AF software (version 1.8.2). Acknowledgements We thank R. Morosoli for providing us with the S. lividans tatC strain. We also thank Kent Loeffler for photography of Arabidopsis plants and R.F. Seipke for providing pRFSRL16. We further thank the Decodon company for support with the Delta 2D software. This work was supported by the CEU project LHSG-CT-2004-005257, the BBSRC through Grant BB/F009224/1 and the MRC via a Senior Non-Clinical Fellowship award to TP. SGM was supported by a PhD studentship funded by the BBSRC and JKF by a joint SCRI/University of Dundee PhD studentship. JKF acknowledges the Society for General Microbiology for funding a research trip to Cornell. Partial support was also provided by the National Research Initiative of the United States Department of Agriculture Cooperative State Research, Education, and Extension Service, Grant number 2008-35319-19202. We thank PCIC, Boyce Thompson Institute (funding sources NSF DBI-0618969 and Triad Foundation) for imaging facilities. References Antelmann, H., Tjalsma, H., Voigt, B., Ohlmeier, S., Bron, S., van Dijl, J.M., and Hecker, M. (2001) A proteomic view on genome-based signal peptide predictions. Genome Res 11: 1484–1502. Bendtsen, J.D., Nielsen, H., Widdick, D., Palmer, T., and Brunak, S. 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